We propose to synthesize a series of polyfunctional organic reagents (PORs) for chemically modifying cell-free human hemoglobins for use as oxygen-carrying blood substitutes in emergency transfusions. These PORs have been specifically designed to perform both infra- and intermolecular hemoglobin cross-linking, with a built-in quality control feature, to yield simultaneously cross-linked and oligomerized hemoglobin products with the desired molecular weights. One of the pressing issues in hemoglobin-based blood substitute research at the present time is to increase the intravascular retention time of the cross-linked hemoglobin, but the current methods employed for polymerization largely yield random, heterogeneous mixtures of products, most of which suffer from problems of auto-oxidation, high oxygen affinity and/or antigenicity. Proposed is a method of controlled oligomerization that yields a specific product of desired molecular weight each time the cross-linking is carried out. We will purify the modified hemoglobins, and investigate their physical, compositional, and functional properties, including molecular weight, methemoglobin levels, storage stability, oxygen affinity (P50), cooperativity (Hill n value), and dependence of P50 on pH (Bohr effect) as well as on anionic species (chloride and phosphate). We will also elucidate the sites of cross-links by a combination of tryptic digestion and MALDI MS analyses. We will then perform extensive animal studies, using rats and rabbits, on the most promising modified hemoglobins to evaluate their intravascular persistence, oxygen transport/delivery, and physiological responses, including hemodynamic reactivity and toxicity.